I. Field of the Invention
The present invention relates generally to the fields of veterinary medicine, reproductive biology and diagnostics. More specifically, the present invention relates to improved methods for accurate pregnancy detection in ungulates (hoofed animals), including ruminants such as bovines (e.g. dairy and beef cattle) and in ovines. The present invention also relates to antibodies, including polyclonal antibodies that have been generated using the PAG-55 enriched protein fraction as an immunogen.
II. Description of the Related Art
Pregnancy diagnosis is an important component in sound reproductive management, particularly in the dairy industry (Oltenacu et al., 1990), where a high proportion of artificial inseminations fail (Streenan and Diskin, 1986). A reliable yet simple pregnancy test for ruminants such as cattle has long been sought. Several procedures are available, including a milk progesterone assay (Oltenacu et al., 1990; Markusfeld et al., 1990), estrone sulfate analysis (Holdsworth et al., 1982; Warnick et al., 1995), rectal palpation (Hatzidakis et al., 1993), ultrasound (Beal et al., 1992; Cameron and Malmo, 1993), and blood tests for pregnancy-specific antigens. Of these, the progesterone milk assay is the most cost effective for the producer (Oltenacu et al., 1990; Markusfeld et al., 1990). Next best is rectal palpation, performed at day 50 post-insemination (Oltenacu et al., 1990).
Even though the prior procedures for pregnancy diagnosis are potentially useful, all have fallen short of expectations in terms of their practical, on-farm use. For example, measurements of milk or serum progesterone around day 18-22 yield unacceptably high rates of false positives (Oltenacu et al., 1990; Markusfeld et al., 1990). The presence of estrone sulfate in urine or serum provides another test, but is only useful after day 100 when concentrations rise (Holdsworth et al., 1982; Warnick et al., 1995).
The discovery of pregnancy-specific protein B (PSP-B) (Butler et al., 1982) provided a new approach to pregnancy diagnosis since it could be detected in the blood of pregnant cows by the fourth week of pregnancy (Sasser et al., 1986; Humblot et al., 1988). Two other groups have developed immunoassays that may be based on an identical or immunologically similar antigen (Zoli et al., 1992a; Mialon et al., 1993; Mialon et al., 1994). In one case, the antigen (Mr˜67 kDa) was called bovine pregnancy-associated glycoprotein (boPAG; now boPAG-1) (Zoli et al., 1991, 1992a, 1992b); in the second, it was designated as pregnancy serum protein 60 (PSP60) (Mialon et al., 1993; Mialon et al., 1994). The term PAG refers generally to pregnancy associated glycoproteins, or pregnancy associated antigens. For a recent review of methods and a characterization of the current members of the boPAGs, see Green et al., Theriogenology 63:1481-1503 (2005), incorporated herein by reference in its entirety.
The immunoassay for PSP-B/boPAG1/PSP60 has several advantages. First, it can detect pregnancy relatively early. Second, interpretation of the assays does not require knowledge of the exact date of service, since boPAG-1 immunoreactive molecules are always present in the maternal serum of pregnant cows by day 28, and concentrations increase as pregnancy advances (Sasser et al., 1986; Mialon et al., 1993; Mialon et al., 1994).
There remain, however, two major disadvantages to this procedure. First, positive diagnosis in the fourth week of pregnancy remains somewhat uncertain because antigen concentrations in blood are low and somewhat variable. Second, boPAG1 concentrations rise markedly at term (Sasser et al., 1986; Zoli et al., 1992a; Mialon et al., 1993) and, due to the long circulating half-life of the molecule (Kiracofe et al., 1993), the antigen can still be detected 80-100 days postpartum (Zoli et al., 1992a; Mialon et al., 1993; Mialon et al., 1994; Kiracofe et al., 1993), compromising pregnancy diagnosis in cows bred within the early postpartum period. Thus, the test can be carried out in dairy cows at day 30 after artificial insemination (AI) only if the AI has been performed at 80 or more days post-partum.
There is a general need for easy, economic, sensitive, non-invasive, and accurate methods and tests for detecting pregnancy analytes in whole blood samples, especially ones that don't require any special equipment or chemicals. These methods would be particularly useful in field or rural settings where laboratory equipment, chemicals, and refrigerated storage are unavailable.
Specifically, there exists a need in the art for a pregnancy test that can be carried out reliably and early in pregnancy which could provide a definitive indication as to whether re-breeding or culling is required. In general, AI (artificial insemination) is successful less than 50% of the time in cattle and the producer must either rely on overt behavioral signals of return to estrus (that are easily missed) or delay re-breeding until pregnancy failure is confirmed by one of the methods described above. Such delays are extremely costly and constitute a major economic loss to the industry.
Specifically, there exists a need in the art for an accurate pregnancy test for ungulates that uses whole blood, which can be easily collected from the animal. Being able to use a whole blood sample with no special equipment or chemical treatment would facilitate reliable and early pregnancy detection allowing efficient action to be taken when re-breeding or culling is required.
Routine immunodetection pregnancy tests involve the detection of an analyte in blood serum. Prior methods for using serum from whole blood for this type of testing involved either additional physical components or matrices such as filters, glass fibers, bibulous agents, paper, fleece, cellulose, wool, asbestos fibers, or required single or multiple centrifugation steps, or required the addition of chemical agents, such as anti-red blood cell antibodies, poly lysine, or lectins.
Whole blood is easily obtained from mammals including farm animals; however, the separation of the interfering constituents such as red blood cells from the desired serum can be problematic in the field, including in farm settings. Thus, there is a need for an easy method that requires no additional filters, matrices, or centrifugation steps, and that allows using whole, clotted blood to detect any desired pregnancy analyte in the serum, using a variety of appropriate tests, such as immunodetection.
Consequently, there is a need for a feasible, economic, sensitive, non-invasive, and accurate pregnancy test for cattle, and other ruminants, that has low levels of false positive results and yet is sensitive enough to detect pregnancy (or more properly the absence thereof) early enough to allow a herd manager to identify non-pregnant (‘open’) cows for re-breeding.